The focus of the studies proposed in this project is the development of rapid diagnostic assays for the detection of human caliciviruses.
In Specific aim 1, we wilt develop broadly-reactive diagnostic reagents for human caliciviruses. Human caliciviruses are divided into two genera, Norovirus and Sapovirus, and within these genera are many genetically and antigenically distinct groups. We have previously identified monoclonal antibodies that recognize epitopes shared among the noroviruses. In addition, we and others have identified blood group antigens involved in binding to norovirus particles. We have recently confirmed the specificity of one of these antigens (Lewis d) for affinity purification of Norwalk virus. These and newly developed monoctonal antibodies and blood group carbohydrates will be characterized for use in the development of rapid, broadly-reactive diagnostic assays.
In Specific aim 2, we will explore new rapid formats to detect human caliciviruses. High affinity, cross-reactive monoclonal antibodies characterized in specific aim 1 will be used in assays that require minimal sample preparation and yield a result in less than 30 minutes. Formats to be evaluated include those that have been used successfully for the detection of other human viruses and include traditional enzyme immunoassays, chromatographic immunoassays, latex agglutination assays, and dot-blot immunoassays. The assays will be tested against a panel of both human and animal caliciviruses and clinical specimens to determine sensitivity, specificity, positive and negative predictive values, and limits of detection. Developed assays will be validated by providing the diagnostic tests to other laboratories (e.g., CDC, state health labs) using RT-PCR assays to evaluate large numbers of specimens for human caliciviruses.
In Specific aim 3, new approaches will be developed to correlate detection of human caliciviruses with their potential infectivity and susceptibility to inactivation. The inability to cultivate human caliciviruses in vitro has hampered assessment of their infectivity. A biosensoring system will utilize green fluorescent protein as a reporter signal that is expressed following the initiation of a viral infection in a transformed cell line. Different disinfection measures will be evaluated for both human and animal caliciviruses and the results will be correlated with those obtained by cell culture for the animal caliciviruses that are cultivatable.
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